Protein folding requires the assistance of folding helpers in vivo. The formation or isomerization of disulfide bonds in proteins is a slow process requiring catalysis. In nascent polypeptide chains the cysteine residues are in the thiol form. The formation of the disulfide bonds usually occurs simultaneously with the folding of the polypeptide, in the endoplasmic reticulum of eukaryotes or in the periplasm of Gram-negative bacteria. Cells contain three types of accessory proteins that function to assist polypeptides in folding to their native conformations: protein disulfide isomerases, propyl cis-trans isomerases, and molecular chaperones.
Protein disulfide isomerase (PDI) is a homodimeric eukaryotic enzyme which catalyzes disulfide interchange reactions. PDI is also thought to be the beta subunit of the heterotetramer prolyl hydrolase, the enzyme that hydroxylates the proline residues in Collagen. PDI appears to belong to a family of closely related proteins which have specific functions. PDI (EC 5.3.4.1), also called S-S rearrangase, catalyzes the rearrangement of both intrachain and interchain disulfide bonds in proteins to form native structures. The reaction depends on sulfhydryl-disulfide interchange, and PDI needs reducing agents or partly-reduced enzyme. A family of PDI-like proteins has been identified in mammals, yeasts, fungi, plants, and Drosophila.
In Drosophila, a PDI precursor was identified by screening a genomic DNA library at reduced stringency hybridization conditions using a rat Phospholipase C alpha cDNA probe. Northern analysis showed that this gene encodes a transcript that is present throughout development, in heads and bodies of adults. The encoded protein contains two domains exhibiting high similarity to thioredoxin, two regions that are similar to the hormone binding domain of human estrogen receptor, and a C-terminal ER-retention signal (KDEL). Overall, this Drosophila PDI gene contains a higher similarity to rat protein disulfide isomerase (53% identical) than to rat Phospholipase C alpha (30% identical) (McKay et al. (1995) Insect Biochem. Mol. Biol. 25:647-654).
Another member of the PDI family is ERp60, a PDI isoform initially misidentified as a phosphatidylinositol-specific phospholipase C. The human and Drosophila ERp60 polypeptides have been cloned and expressed. These two ERp60 polypeptides are similar to human PDI within almost all their domains, the only exception being the extreme C-terminal region. Coexpression in insect cells of the human or Drosophila ERp10 with the alpha subunit of human propyl 4-hydrolase does not result in tetramer formation or prolyl 4-hydroxylase activity in the cells. This lack of tetramer formation is not only due to the differences in the C-terminal region since no prolyl 4-hydroxylase tetramer is formed when a human ERp60 hybrid containing the C-terminal region of the human PDI polypeptide is used (Koivunen et al. (1996) Biochem. J. 316:599-605). The 5′ flanking region of the ERp60 gene has no TATAA box or CCAAT motif but contains several potential binding sites for transcription factors. The highest levels of expression of the human ERp60 mRNA are found in the liver, placenta, lung, pancreas, and kidney, and the lowest in the heart, skeletal muscle, and brain. The ERp60 gene has been mapped by fluorescence in situ hybridization to 15q15, a different chromosome than where the human PDI and thioredoxin genes are found (Koivunen et al. (1997) Genomics 42:397-404).
Full-length cDNA clones encoding two members of the mice PDI family have been cloned, sequenced, and expressed (ERp59/PDI and ERp72). ERp59/PDI has been identified as the microsomal PDI. The ERp72 amino acid sequence shares sequence identity with ERp59/PDI at three discrete regions, having three copies of the sequences that are thought to be the CGHC-containing active sites of ERp59/PDI. ERp59/PDI has the sequence Lys-Asp-Glu-Leu at its COOH terminus while ERp72 has the related sequence Lys-Glu-Glu-Leu (Mazzarella et al. (1990) J. Biol. Chem. 265:1094-1101). A cDNA clone containing sequence similarity to the mammalian lumenal endoplasmic reticulum protein ERp72 has been isolated from an alfalfa (Medicago sativa L.) cDNA library by screening with a cDNA encoding human PDI. The polypeptide encoded by this cDNA possesses a putative N-terminal secretory signal sequence and two regions identical to the active sites of PDI and ERp72. This protein appears to be encoded by a small gene family in alfalfa, whose transcripts are constitutively expressed in all major organs of the plant. In alfalfa cell suspension cultures, ERp72 transcripts are induced by treatment with tunicamycin, but not in response to calcium ionophore, heat shock or fungal elicitor (Shorrosh and Dixon (1992) Plant J. 2:51 -58).
Another member of the PDI family is ERp5. The amino acid sequence deduced from this cDNA insert contains two copies of the 11-amino-acid sequence Val-Glu-Phe-Tyr-Ala-Pro-Trp-Cys-Gly-His-Cys. Duplicate copies of this sequence are found in the active sites of rat and human PDI and in Form I phosphoinositide-specific phospholipase C. Genomic sequences similar to the cDNA clone are amplified 10-20-fold in hamster cells selected for resistance to increasing concentrations of hydroxyurea, a phenomenon observed earlier with cDNA clones for the M2 subunit of ribonucleotide reductase and ornithine decarboxylase. RNA blots probed with ERp5 cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells (Chaudhuri et. al. (1992) Biochem. J. 281:645-650).
A PDI-like protein from Acanthamoeba castellanii contains two highly conserved thioredeoxin-like domains, each about 100 amino acids. However, the A. castellanii PDI-like protein differs from other members in many aspects, including the overall organization and isoelectric point. Southern and Northern analyses demonstrate that the PDI-like protein is encoded by a single-copy gene which is transcribed to generate a 1500-nucleotide MRNA (Wong and Bateman (1994) Gene 150:175-179).
The Chlamydomonas RB60 gene encodes a chloroplast-localized PDI which is involved in the redox-regulated binding of chloroplast poly(A)-binding protein to the 5′-leader region of psbA MRNA. Protein disulfide isomerase RB60 regulates chloroplast translational activation (Kim and Mayfield (1997) Science 278:1954-1957).
High level gene expression does not always lead to corresponding high level secretion of heterologous proteins. The rate limiting step has been shown, in many cases, to be the processing and exit of the protein from the endoplasmic reticulum. Proteins or peptides with high levels of disulfide bonds can be adversely affected during expression. Therefore, coexpression and/or overexpression of PDIs could significantly enhance expression levels of many heterologous proteins. An example would be the coexpression of PDIs with insect-selective neurotoxins, since many of these are highly enriched in cysteines and feature multiple disulfide bonds.
Protein disulfide isomerases have been described in alfalfa (2 genes and one probable PDI P5 homolog), barley (2 genes, and one probable PDI PS homolog), maize, wheat, tobacco, and castor bean. In addition, based on sequence similarity to other known PDIs, two putative protein disulfide isomerases have been identified in Arabidopsis. Included in this application are corn, and soybean ESTs with sequence similarities to protein disulfide isomerase precursor. The corn sequences included share no similarity with the known maize PDI. Also included are corn, balsam pear, soybean, and the wheat ESTs with sequence similarities to RB60. Presently there are no plant RB60-homologs in the public domain. Overexpression of any of these PDIs together with another foreign protein will result in an increased yield of secreted, active foreign protein due to proper folding of the foreign protein.
Present in the NCBI database are corn and soybean sequences with similarities to the polynucleotides included in the present application. These ESTs have NCBI General Identifier NOs:4289796, 4827500, 5124153, 5325044, 5361231, 5525515, 5597319, 5650368, 5688597, 5714111, 5770161, and 5804735.